The broad, long-term objective of the proposed project is to enable mass-spectrometry-based protein research. The project will develop algorithms and software for de novo peptide sequencing by tandem mass spectrometry, taking advantage of two currently unexploited information channels: protein family homology, and multiple complementary spectra of the same peptide. Homology will allow the de novo sequencing program to limit attention to peptides fitting certain motifs or constraints. With strict constraints, the program would search only the most likely mutations of known sequences, but with loose constraints the program would search as widely as current de novo sequencing programs. Complementary spectra, for example, one CID (collision-induced dissociation) and one ETD (electron-transfer dissociation) spectrum, give more complete fragmentation, and more importantly, help the program recognize which peaks correspond to which ion types, for example, helping to distinguish b-ions from y-ions. New software produced by the project will be put to immediate use in sequencing antibodies and toxins. The specific aims of the project are: homology-guided generation of candidate peptides, utilization of multiple and complementary spectra (for both candidate generation and scoring), and application and deployment of the software for sequencing polyclonal serum antibodies and complex mixtures of snail and spider toxins. These are high-value targets because the antibodies are reactive to HIV, and the toxins are valuable both as probes to study ion channels and also as pharmaceutical leads. If the project achieves its aims, exact sequencing of unknown peptides will become much easier than it is today, laboratories worldwide will take advantage of the new data acquisition and analysis strategies, and biomedical discovery will be greatly accelerated. PUBLIC HEALTH RELEVANCE: The proposed project is important to public health because it will enable exact identification of unknown, unsequenced proteins, including spider and snail toxins and polyclonal antibodies captured from human serum. Snail toxins are already the basis for FDA-approved analgesics. Captured antibodies could enable antibody treatments for HIV infection and other diseases. .